The abuse of illicit drugs such as cocaine, marijuana and heroin remains a critical public health concern throughout the country and is associated with staggering economic and social consequences. Cocaine remains the most frequently recorded illicit drug in emergency hospital visits and the leading cause of drug- related deaths in the United States. Since as high as 23% of fatally injured drivers were found positive results in drug tests, the Office of National Drug Control Policy emphasizes the major concern of drugged driving in the United States. Urinalysis is the most common type of cocaine test, though urine collection has challenged its practicality. There are other biological specimen-based cocaine tests that have been explored such as blood and saliva tests but are not practical since blood samples need high-level medical administrators and specialized analytic equipment. Another alternative is the minimally invasive diagnosis through skin using surface modified microneedles (MNs). However, since concentrations of most blood biomarkers in the skin are too low to be captured by the MN array, the test suffers from low sensitivity and high variation. Here we propose a novel probe-MN-based assay in conjunction of using brief laser illumination on a tiny skin area to accumulate blood biomarker, such as cocaine into the upper dermis. This sample accumulation method enables the biomarker to be readily detected and quantified in situ using the probe-MNs inserted into the laser- treated skin. The objective for the STTR Phase I is to characterize and validate sensitivity and specificity of the probe-MN array in vitro and in vivo, and compare detection reliability with conventional analysis of cocaine in plasma and in urine samples. The specific aims are as follows: Aim 1: Determine optimal design of cocaine-specific binding MN probe and characterize cocaine assay in vitro. We will develop a protocol of surface modification of MNs with Cy3-labeled cocaine-specific aptamer and Cy5- labeled neutralizer. Length of the MNs ranging from 50 - 300um will be optimized based on iterative feedback from in vivo test. Sensitivity, specificity, and response time of the MNs array will be evaluated by in vitro tests. Aim 2: Demonstrate the feasibility and consistence of upper dermal cocaine detection in mice and rats. The cocaine-specific MN probe will be evaluated in mice and rats to demonstrate feasibility of the proposed minimally invasive MN-based cocaine detection in conjunction of using a brief illumination of a clinically safe dose of laser on the skin. Sensitiviy, specificity, and reliability will be determined with respect to conventional assays of plasma and urinalysis of cocaine. The ultimate goal of the entire STTR project is to develop and commercialize the minimally invasive, sample- free, in situ cocaine detection toolkit consisting of device and cocaine-specific probe-MN arrays for prompt onsite cocaine detection in humans in community, work places, or emergency rooms and clinics.